Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

Radioprotection of human cell nuclear DNA by polyamines: radiosensitivity of chromatin is influenced by tightly bound spermine

The polyamines putrescine (PUT) and spermine (SPM) were examined for their ability to protect human cell DNA against the formation of radiation-induced double-strand breaks (DSBs). As observed previously, under conditions where polyamines were shown to be almost completely absent, association with nuclear matrix protein into a nucleoid, and organization into chromatin structure, protected DNA from induction of DSBs by factors of 4.5 and 95, respectively. At concentrations below 1 mM, PUT or SPM provided equivalent levels of protection to deproteinized nuclear DNA, consistent with their capacity to scavenge radiation-induced radicals. At constant ionic strength, 5 mM SPM protected deproteinized DNA and nucleoid DNA and DNA in nuclear chromatin by factors of 100 and 26, respectively. At 5 mM, SPM provided 15 times greater protection of deproteinized DNA than did PUT. Under physiologically relevant conditions, 5 mM SPM protected DNA in the intact nucleus from the induction of DSBs by a factor of 2 relative to DNA in the absence of SPM. Studies of SPM binding during cellular fractionation revealed that a significant fraction of the cellular SPM is tightly bound in the nucleus but can be removed by extended washing. Thus the association of SPM with nuclear chromatin appears to be a significant contributor to the resistance of the cell's DNA to the induction of DSBs.     

Inhibitor effect of polyamines on reduction of cytochrome C by superoxide anion

The interaction of polyamines with enzymatically generated free radicals was investigated. The superoxide anion (O-2) was generated in vitro using the xanthine oxidase-hypoxanthine system. Our results show that spermidine or spermine at different concentrations (20-200 mM) inhibit the reduction of cytochrome c; the highest levels of inhibition were obtained adding 200 mM spermidine or spermine. Putrescine (200 mM) affected the reduction of cytochrome c very little.     

Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80 degrees C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against gamma-radiation-induced death.     

The influence of spermidine (polyamines) on DNA depurination in vitro

The influence of the spermidine, spermine and putrescine on the DNA depurination rate was studied. These polyamines protect DNA against depurination. The rate of Col EI DNA depurination at pH 4.3 was decreased over 10-fold by addition of 10 mM polyamines.     

Nuclear aggregates of polyamines in a radiation-induced DNA damage model

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs–pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA.     

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe(3+) to Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-ethylene diaminetetraacetic acid, Fe(3+)-ethylene diaminetetraacetic acid systems with and without H(2)O(2); (3) protect DNA from damage caused by Cu(2+)-H(2)O(2), and Fe(2+)-H(2)O(2) with and without ascorbic acid; (4) inhibit H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1mM reduced 1.8+/-0.3 and 2.5+/-0.1 nmol of Fe(3+) ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe(3+)-ethylene diaminetetraacetic acid. An 10mM spermine and spermidine decreased CuSO(4)-H(2)O(2)-ascorbic acid- and FeSO(4)-H(2)O(2)-ascorbic-induced DNA damage by 73+/-6, 69+/-4% and 90+/-5, 53+/-4%, respectively. They did not protect DNA from CuSO(4)-H(2)O(2) and FeSO(4)-H(2)O(2). Spermine apparently increased the CuSO(4)-H(2)O(2)-dependent injury to DNA. Polyamines attenuated H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10mM spermine or spermidine was attenuated by 85.3+/-1.5 and 87+/-3.6%. During 20 min incubation 1mM spermine or spermidine decomposed 8.1+/-1.4 and 9.2+/-1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H(2)O(2).     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

Effect of polyamines on ADP-ribosylation by chick-embryo-liver nuclei

Effects of polyamines on poly(ADP-ribose) formation and DNA synthesis in the chick-embryo-liver nuclei were investigated. When 14-day chick-embryo-liver nuclei were incubated with [3H]NAD in the presence of 1 mM spermine, 2.5 mM spermidine, or 3.5 mM putrescine, a 9-fold increase in poly)ADP-ribose) formation was observed. Nuclei treated with nuclease showed high poly(ADP-ribose) synthetase activity as spermine-treated nuclei. However, no further increase in the polymer formation by polyamines was detected in the nuclease-treated nuclei. We found that an increase in the polymer formation by spermine was the result of an increase in both chain length and chain number of the polymer at 2.3- and 6-fold, respectively. The major ADP-ribosylated proteins were determined as two non-histone proteins of Mr 130 000 and 70 000. The experiment of DNA synthesis with nuclei ADP-ribosylated in the presence of spermine showed a 7-fold increase in [3H]dTMP incorporation into the acid-inaoluble fraction. A similar stimulation was also found with nuclei treated with other polysmines, spermidine and putrescine, in the presence of NAD. These results indicate that DNA synthesis in growing tissues containing polyamines at high levels, such as is the case with tumors and the fetus, is stimulated by polyamine-mediated ADP-ribosylation of the nuclear proteins.     

Yield of DNA strand breaks after base oxidation of plasmid DNA

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

Copper-catalyzed DNA damage by ascorbate and hydrogen peroxide: kinetics and yield

Time profiles for degradation of DNA via reaction of H2O2 with the DNA-Cu+ complex were analyzed over a wide range of concentrations of the components. The yield of DNA damage per H2O2 molecule is 10 times lower than that obtained with gamma-radiolytically generated .OH radicals. The observations can be explained by a model in which H2O2 reacts, slowly on the one hand with DNA-Cu+ by formation of toxic .OH radicals immediately at the DNA and faster on the other hand with Cu+ in the bulk solution by formation of less toxic Cu(III) intermediates.     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Activation of phosphatidylinositol-4-phosphate kinase in rat liver plasma membranes by polyamines

The formation of phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous substrate in rat liver plasma membranes was stimulated approximately 3-fold by 1 mM spermine, with half-maximal effect at 0.2 mM polyamine. This effect of spermine was due to enhancement of phosphatidylinositol-4-phosphate kinase activity rather than to a decrease in degradation of PIP2 formed or the substrate phosphatidylinositol 4-phosphate (PIP). The stimulation of phosphatidylinositol-4-phosphate kinase by spermine decreased to half at physiological ionic strength, and was not affected appreciably by variations in the concentration of ATP and MgCl2. Among several di- and polyamines only spermine and spermidine were effective. Although spermine may cause aggregation of membrane vesicles, thereby potentially increasing substrate availability for phosphatidylinositol-4-phosphate kinase, our results do not support such an explanation for the enhancement in enzyme activity. Phosphatidylinositol kinase activity, contrary to phosphatidylinositol-4-phosphate kinase, was not stimulated appreciably by spermine.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Polyamines protect against DNA strand breaks and aid cell survival against irradiation in Escherichia coli

Polyamines protected plasmid DNA strand breaks in vitro and aided the cell survival against irradiation in polyamine-deficient Escherichia coli mutant strain. DNA strand breaks were prevented 4–6 fold more by spermidine and spermine than by putrescine and cadaverine in the dithiothreitol/Fe(III)/O2 system. After UV-irradiation, the protection of DNA strand breaks by spermine and spermidine was twice as effective as that by putrescine and cadaverine. Survivability of polyamine-deficient Escherichia coli mutant cells grown in the medium containing putrescine and spermidine was 2.4- and 3.0-fold as high as in polyamine-depleted medium at a dose of 60 and 40 J/m2. After -irradiation to a dose of 80 Gy, cell survivals of a mutant strain were significantly increased to 7.7- and 23.8-fold by putrescine and spermidine, respectively. These results implicate the possibility that polyamines play a potent role in the protection of DNA or cell damage by radiation. © Rapid Science Ltd. 1998     

Fourier transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines

Biogenic polyamines putrescine, spermidine, and spermine are essential molecules for proliferation in all living organisms. Direct interaction of polyamines with nucleic acids has been proposed in the past based on a series of experimental evidences, such as precipitation, thermal denaturation, or protection. However, binding between polyamines and nucleic acids is not clearly explained. Several interaction models have also been proposed, although they do not always agree with one another. In the present work, we make use of the Raman spectroscopy to extend our knowledge about polyamine-DNA interaction. Raman spectra of highly polymerized calf-thymus DNA at different polyamine concentrations, ranging from 1 to 50 mM, have been studied for putrescine, spermidine, and spermine. Both natural and heavy water were used as solvents. Difference Raman spectra have been computed by subtracting the sum of the separated component spectra from the experimental spectra of the complexes. The analysis of the Raman data has supported the existence of structural specificities in the interactions, at least under our experimental conditions. These specificities lead to preferential bindings through the DNA minor groove for putrescine and spermidine, whereas spermine binds by the major groove. On the other hand, spermine and spermidine present interstrand interactions, whereas putrescine presents intrastrand interactions in addition to exo-groove interactions by phosphate moieties.